Molecular diagnostics aims at the rapid detection of minute amounts of pathogens (typically bacteria) in samples such as blood. However, blood is a complex matrix and comprises white blood cells (leukocytes) for the adaptive immune system, red blood cells (erythrocytes) for oxygen transport, and platelets (thrombocytes) for wound healing. This composition complicates the direct detection of pathogens in samples such as whole blood, which contain a high amount of cellular material such as DNA and proteins.
Therefore, methods for enriching pathogens while removing blood cellular material from a blood sample are required.
In a known method, pathogens are collected onto a filter by filtration of large volume blood samples after a selective lysis of blood cells.
In the filtration step, inhibitory components such as DNA or proteins are removed while the pathogens are retained onto the filter.
The International patent application WO-A-2011/070507 discloses a method for selective lysis of eukaryotic cells within a sample containing or suspected to contain micro-organisms, wherein a detergent and a buffer are added to said sample to obtain a solution which is further incubated for a time period sufficiently long enough to lyse the eukaryotic cells. This method permits processing of blood samples having a volume of the order of 5 to 10 ml by lysing the white and red blood cells in the sample, degrading the white blood cell DNA while a substantial part (i.e. more than 20%, 40%, 60%, 80%, 90% or even more than 95%) of the micro-organisms remains alive, or if killed by the treatment, still comprise the bacterial DNA within the cell wall. Said micro-organisms can subsequently be enriched by a filtration step so that inhibitory components such as DNA or proteins are removed while the pathogens are retained onto the filter with their bacterial DNA within the cell wall.
It is important that all the available volume of sample is filtered to collect a maximum of micro-organisms onto the filter. However, when the method is applied, the filter usually gets clogged before the sample is completely filtered, due, for example, to the presence of proteins in the sample that are also retained by the filter. This clogging leads to an abortion of the filtration thus stopping the whole processing. Therefore, the existing method does limit the quantity of sample that can be processed on a filter.